This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We previously reported comparative studies of the protective efficacy of different "prime-boost" immunization regimens consisted of priming with recombinant vaccinia or DNA vectors expressing HIV-1 SF162 Env or SIVmac239 Gag/Pol, followed by boosting with DNA, protein;or heterologous viral vector expressing the same antigens. Results showed that, regardless of the priming immunogen, animals boosted with protein vaccines had significantly higher antibody titers, including homologous neutralization antibodies (NtAb). After SHIVSF162P4 challenge, significant reduction of mean plasma viral load was observed in all immunization groups. An inverse correlation (Spearman's r = -0.819, p0.0001) was observed between NtAb titer on the day of challenge and peak plasma viral load after challenge. Five of 12 animals boosted with proteins showed high NtAb titer and no detectable viremia after challenge, consistent with protection from infection. We have also extended the comparative study to include protein priming, followed by boosting with DNA, protein or viral vector. After two immunizations with protein vaccines, high level of virus-specific antibody responses, including homologous NtAb, were generated. However, further immunizations with DNA or viral vectors showed little or no boosting effects. Following intrarectal SHIV162 P4 challenge, all but one animal in each of the protein-DNA or protein-protein prime-boost groups were infected. These results further support the important role of priming in the prime-boost immunization in the generation of protective immunity against primate lentivirus infection. To further define the nature of neutralizing antibody responses in these animals, PBMC were sent to Dr. James Robinson at Tulane University to isolate neutralizing monoclonal antibodies against HIV-1. Preliminary data from Dr. Robinson indicate that some of these antibodies may recognize conformation-dependent epitopes against HIV-1 envelope. Further characterization of these antibodies is in progress.